Noninfected Rats Research Articles

Exploring Antigenic Properties and Immune Responses in Bufo himalayanus: Implications for Conservation and Biodiversity Protection

Background: The objective of the study is to investigate antigenic property of soluble antigens of Nematodes Cosmocercoides dukae isolated from infected Bufo himalayanus (Class Amphibia, order: Anura) and to investigate the nature of protective immune response in laboratory rats. Bufo himalayanus is a species found in the districts of Darjeeling, Sikkim and adjoining hills of the sub-Himalayan belt, at an altitude of 6000 ft. above the sea level and known to be heavily infested by natural gut dwelling nematode infections. The present study is a small step towards protection and conservation of this amphibian species.
 Results: study of morphology of the nematode parasite confirmed it to be Cosmocercoides dukae. A crude whole body homogenate (antigenic protein) of the parasite was injected in healthy non-infected rats, spleen histo-architecture of treated rats differed from control rats. Humoral immune response assay by Gel Diffusion Precipitin (GDP) Test revealed that the parasite antigen invoked very little to no response. Study of the crude nematode antigenic protein extract concentration was 6.09 mg/ml and protein profile assay by SDS-PAGE showed seven major and several minor bands.
 Discussion: Though humoral immune response assay of parasite in experimental rats evoked very little or no immune response, this would not mean absence of antibodies, rather the antibody titers were presumably very low. It is to be noted that spleen of treated rats showed a distinctive histological feature. GDP test with infected toad serum also showed negative result. But protein profile assay of antigenic protein by SDS-PAGE showed many bands of proteins, which could be isolated and tested for further characterization.
 Conclusion: In order to maintain the Himalayan biodiversity, this amphibian species needs protection from such natural infective nematode parasites. If we are able to focus any light to this area, this knowledge might help the future research workers to plan for the protection of this species.

Gene therapy for alopecia in type II rickets model rats using vitamin D receptor-expressing adenovirus vector

Type II rickets is a hereditary disease caused by a mutation in the vitamin D receptor (VDR) gene. The main symptoms of this disease are bone dysplasia and alopecia. Bone dysplasia can be ameliorated by high calcium intake; however, there is no suitable treatment for alopecia. In this study, we verified whether gene therapy using an adenoviral vector (AdV) had a therapeutic effect on alopecia in Vdr-KO rats. The VDR-expressing AdV was injected into six 7-week-old female Vdr-KO rats (VDR-AdV rats). On the other hand, control-AdV was injected into 7-week-old female rats (control-AdV rats); non-infected Vdr-KO rats (control rats) were also examined. The hair on the backs of the rats was shaved with hair clippers, and VDR-AdV or control-AdV was intradermally injected. Part of the back skin was collected from each rat after AdV administration. Hair follicles were observed using hematoxylin and eosin staining, and VDR expression was examined using immunostaining and western blotting. VDR-AdV rats showed significant VDR expression in the skin, enhanced hair growth, and low cyst formation, whereas control-AdV and non-infected rats did not show any of these effects. The effect of VDR-AdV lasted for nearly 60 days. These results indicate that gene therapy using VDR-AdV may be useful to treat alopecia associated with type II rickets, if multiple injections are possible after a sufficient period of time.

Potential therapeutic effect of platelet-rich plasma and albendazole on the muscular phase of experimental Trichinella spiralis infection

Trichinellosis is a food-borne parasitic infection causing muscle damage. This study aimed to detect the potential therapeutic effect of platelet-rich plasma (PRP) alone or in combination with albendazole (ALB) on the muscular phase of experimental Trichinella infection in rats. The study was conducted on 70 rats divided into four main groups: healthy non-infected non-treated rats, non-infected rats treated with PRP, infected untreated rats (seven rats in each group), and an infected group of 49 rats. The infected group was further subdivided based on the drug therapy received. The effects of drug therapy were evaluated using parasitological and histopathological analyses.The percent reduction in the number of Trichinella spiralis larvae per gram of muscle in the PRP-treated groups (one, two, and three doses) was 43.1%, 78.8%, and 86.1%, respectively. Groups treated with combined therapy of ALB & PRP (one, two, and three doses) showed overall reduction percentages of 87.7%, 90.9% and 95.2%, respectively. In contrast, the ALB-treated group showed a 69.4% reduction. All results of the abovementioned groups were statistically significant compared to the control-infected non-treated group.The findings of the histopathological analysis were consistent with the parasitological results. Groups receiving combined therapy showed the most significant improvement in terms of the degree of inflammation and fibrosis. It can be concluded that PRP has a modulatory effect on the pathology caused by T. spiralis larvae in the muscular phase of trichinellosis.To our knowledge, this is the first study to investigate the effect of PRP on the muscular phase of T. spiralis infection.

Wound-Healing and Antibacterial Activity of the Quercetin–4-Formyl Phenyl Boronic Acid Complex against Bacterial Pathogens of Diabetic Foot Ulcer

Complications of diabetic foot can be prevented using a naturally occurring, efficient, and newly synthesized antimicrobial agent. The purpose of the study was to improve wound healing and antibacterial effects of quercetin and its esterified complex with 4-formyl phenyl boronic acid (4FPBA–Q) compared with phenytoin. The formation of the 4FPBA–Q complex was confirmed by thin-layer chromatography (TLC), Fourier transform infrared (FTIR) spectroscopy, and mass spectrometry (MS). The prepared 4FPBA–Q complex was used against Gram-positive bacteria along with Gram-negative bacteria, and more than 2-fold decrease in minimum inhibitory concentrations (MIC) was observed compared to pure quercetin. Scanning electron microscopic images of Pseudomonas aeruginosa and Staphylococcus aureus showed their complete destruction after incubation with the 4FPBA–Q complex even after 3 h. Interestingly, wound-healing properties of the 4FPBA–Q complex in infected diabetic rats increased from 64 to 99% as compared to phenytoin, which were increased from those of noninfected diabetic rats. Furthermore, histopathological evaluations showed significantly enhanced wound healing, re-epithelialization, fibroblasts, and angiogenesis in wounds of diabetic rats after 10 days. Conclusively, reduction in the primary irritation index (PDII) and improved antibacterial and wound-healing properties render the 4FPBA–Q complex ideal for diabetic foot ulcer treatment.

Infection with intestinal helminth (Hymenolepis diminuta) impacts exploratory behavior and cognitive processes in rats by changing the central level of neurotransmitters.

Parasites may significantly affect the functioning of the host organism including immune response and gut-brain-axis ultimately leading to alteration of the host behavior. The impact of intestinal worms on the host central nervous system (CNS) remains unexplored. The aim of this study was to evaluate the effect of intestinal infection by the tapeworm Hymenolepis diminuta on behavior and functions of the CNS in rats. The 3 months old animals were infected, and the effects on anxiety, exploration, sensorimotor skills and learning processes were assessed at 18 months in Open Field (OF), Novel Object Recognition (NOR) and the Water Maze (WM) tests. After completing the behavioral studies, both infected and non-infected rats were sacrificed, and the collected tissues were subjected to biochemical analysis. The levels of neurotransmitters, their metabolites and amino acids in selected structures of the CNS were determined by HPLC. In addition, the gene expression profile of the pro- and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) was evaluated by Real-Time PCR to determine the immune response within the CNS to the tapeworm infection. The parasites caused significant changes in exploratory behavior, most notably, a reduction of velocity and total distance moved in the OF test; the infected rats exhibited decreased frequency in the central zone, which may indicate a higher level of anxiety. Additionally, parasite infestation improved spatial memory, assessed in the WM test, and recognition of new objects. These changes are related to the identified reduction in noradrenaline level in the CNS structures and less pronounced changes in striatal serotonergic neurotransmission. H. diminuta infestation was also found to cause a significant reduction of hippocampal expression of IL-6. Our results provide new data for further research on brain function during parasitic infections especially in relation to helminths and diseases in which noradrenergic system may play an important role.

English

Diabetic ulcers are a severe complication in patients with diabetes that often lead to amputation. The etiology of diabetes-induced wound healing impairment is multi-faceted. Persistent inflammation and deficient tissue regeneration have been suggested to impair wound healing in diabetes patients. Although new therapeutic approaches have been developed, many chronic wound treatments remain unsatisfactory, and more promising therapeutics are urgently needed. We thus developed diabetic wounding models with or without bacterial infection in rats that are optimized for testing a traditional Chinese medicine-Zizhu ointment. We observed increased proinflammatory markers and decreased angiogenesis in both non-infected and infected diabetic wounded rats. These changes were normalized by Zizhu ointment. Additionally, activation of Phosphatidylinositol-3-kinase (PI3K), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB) were observed in diabetic wounded rats, whereas Zizhu ointment significantly inhibited activation of these proteins. Our results suggest that Zizhu ointment promotes wound healing by inhibiting the inflammatory response. Key words: Zizhu ointment, diabetic wounding, inflammation, angiogenesis, nuclear factor-kappa B (NF?κB).

MiR-141-3p inhibits the activation of astrocytes and the release of inflammatory cytokines in bacterial meningitis through down-regulating HMGB1

BackgroundBacterial meningitis (BM) is a serious infectious disease of the central nervous system that often occurs in children and adolescents. Many studies have suggested that microRNAs (miRNAs) are involved in BM. This study aimed to address the effects of miR-141-3p on astrocyte activation and inflammatory response in BM through HMGB1. MethodsThe 3-week-old rats were injected with Streptococcus pneumoniae (SP) into the lateral ventricle to establish a BM model. Loeffler scoring method was used to evaluate the recovery of neurological function. Brain pathological damage was observed by hematoxylin and eosin (H&E) staining. Primary astrocytes were isolated from brain tissues of BM or non-infected SD rats. The levels of TNF-α, IL-1β, and IL-6 in brain tissues and astrocyte culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between miR-141-3p and HMGB1 was tested using dual-luciferase reporter assay. The expression of miR-141-3p, HMGB1, and the astrocytic marker glial fibrillary acidic protein (GFAP) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blotting. Methylation-specific PCR (MSP) analysis was performed to measure the methylation status of miR-141 promoter. ResultsThe results showed that lower Loeffler scores were exhibited in rats with BM. The subarachnoid space of brain tissues of BM rats was widened, and obvious inflammatory cells were observed. miR-141-3p expression was reduced in BM rats and SP-treated astrocytes. Additionally, we found that overexpression of miR-141-3p led to the downregulation of HMGB1, GFAP, and inflammatory cytokines (TNF-α, IL-1β, and IL-6) in astrocytes. Furthermore, the results of dual-luciferase reporter assay confirmed that miR-141-3p directly targeted HMGB1. Overexpression of miR-141-3p inhibited the levels of GFAP, TNF-α, IL-1β, and IL-6 in astrocytes, which was eliminated by the up-regulation of HMGB1. The results of MSP analysis indicated that miR-141 promoter was highly methylated in brain tissues and astrocytes. DNMT1 was involved in the methylation of miR-141 promoter in BM. ConclusionThe present study verified that miR-141-3p affected inflammatory response by suppressing HMGB1 in SP-induced astrocytes and BM rat model.

Alkaloidal fraction of Diospyros mespiliformis protect against Trypanosoma evansi-mediated haematological and hepatic impairment in infected rats

The present study investigated the antitrypanosomal activities of crude and an alkaloidal fraction of Diospyros mespiliformis in Trypanosoma evansi - infected rats. A total of twenty-one (21) rats were divided into seven (7) groups of three (3) rats each. Groups 1-6 were infected with the T. evansi parasite and were treated with 100 and 200 mg/kg BW crude extract, 200 and 400 mg/kg BW of the alkaloid fraction of D. mespiliformis, 3.5mg/kg of berenil (standard control) and 0.2 mL/kg BW of normal saline (negative control) respectively. Group 7 serves as the normal control (non-infected and non-treated) rats. Results revealed that the crude extract at 400 mg/kg BW and alkaloid fraction at all doses tested (100 and 200 mg/kg BW) significantly (P<0.05) increased the RBC, PCV, MCH, MCHC, WBC, and reduced the elevated bilirubin when compared with the untreated control. The extract also significantly increased the reduced total proteins. In conclusion, an alkaloid from Diospyros mespiliformis ameliorative effect on T. evansi-induced biochemical and hematological alterations in rats, thus could be considered a novel agent for the development of a new drug against trypanosomiasis

Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats.

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague-Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 106, 107 or, 108 CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 107 or 108 CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 10³ CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16. h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Efek Proteksi dari Terapi Oksigen Hiperbarik terhadap Ekspresi Bcl-2 Miometrium Rattus norvegicus Bunting yang Terinfeksi oleh Tachyzoite Toxoplasma gondii

Hyperbaric Oxygen Therapy (HBOT) can increase oxygen delivery to tissues and stimulate the formation of H2O2 as a secondary messenger for phosphorylation of nuclear factor kappa beta (NF-kB) which plays an important role in the transcription of the anti apoptotic gene. This study aimed to determine the effects of Hyperbaric Oxygen Therapy (HBOT) in enhancing the expressions of Bcl-2 in the myometrium of pregnant rats infected by Toxoplasma gondii. This study was an experimental study with a randomized control group of post-test only and designed by 37 pregnant Rattus norvegicus Sprague Dawley. Randomly, the rats were divided into four groups. Group A is infected pregnant rats that exposed by 10 sessions of HBOT 2.4 ATA in 3x30 minutes. Group B is non-infected pregnant rats and exposed by 10 sessions of HBOT 2.4 ATA in 3x30 minutes. Group C is infected pregnant rats without any exposure. Group D is non-infected pregnant rats without any exposure. Each infected pregnant rat was given a 103 tachyzoite of T.gondii by intraperitoneal injection. Bcl-2 expressions were measured through immunohistochemistry. All data were analyzed using ANOVA test through SPSS 21 program application. There was a significant difference in Bcl-2 expression between Group A and Group C because p<α (p<0.017). HBOT can increase the expression of Bcl-2 from infected and not infected rat myometrium, in the provision of HBOT 2.4 ATA for 3x30 minutes, twice a day for 5 days.

Interactions between local renin angiotensin system and nitric oxide in the brain of Trypanosoma cruzi infected rats

Chagas’ disease (CD) is a zoonosis caused by the protozoan Trypanosoma cruzi. Besides being an important cause of cardiomyopathy, central nervous system (CNS) manifestations have also been reported in CD. Renin-Angiotensin System (RAS) plays a pathophysiological role in several brain disorders such as cerebrovascular and neurodegenerative diseases. A link between RAS and nitric oxide (NO) pathways has been described in CNS. For instance, Angiotensin-(1–7) increases NO expression in the brain, which may, in turn, help to control parasite load in response to T. cruzi infection. Herein, we investigated the levels of RAS components in the brain cortex in acute T. cruzi infection and the effect of L-NAME administration, an inhibitor of the enzyme NO synthase, in CNS infection and in RAS molecules. Male Holtzman rats were inoculated intraperitoneally with T. cruzi Y strain and received L-NAME or tap water from one day before the infection until 13 days post infection (dpi). Parasitemia was evaluated on alternate days from day 3 post-infection until day 13 in both T. cruzi infected groups. Histopathological analysis of the brain cortex was also performed. Brain cortex was collected from non-infected (controls) and infected rats at 13 dpi for RAS components assessment. Infected rats receiving L-NAME presented higher parasitemia, brain parasitism and inflammation compared with non-treated infected animals. The administration of L-NAME significantly decreased the levels of Angiotensin I Converting Enzyme 2 (ACE2). In conclusion, we provided preliminary evidence of the interaction between RAS and NO during the acute phase of T. cruzi infection.

Inbred Rats as a Model to Study Persistent Renal Leptospirosis and Associated Cellular Immune Responsiveness.

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Rats are regarded as one of the most significant reservoir hosts of infection for human disease, and in the absence of clinical signs of infection, excrete large numbers of organisms in their urine. A unique biological equilibrium exists between pathogenic leptospires and reservoir hosts of infection, but surprisingly, little is known concerning the host's cellular immune response that facilitates persistent renal colonization. To address this deficiency, we established and applied an immunocompetent inbred rat model of persistent renal colonization; leptospires were detected in urine of experimentally infected rats by 3 weeks post-infection and remained positive until 8 weeks post-infection. However, there was little, if any, evidence of inflammation in colonized renal tubules. At 8 weeks post-infection, a robust antibody response was detected against lipopolysaccharide and protein outer membrane (OM) components. Purified B and T cells derived from the spleen of infected and non-infected rats proliferated in response to stimulation with 0.5 μg of OM fractions of Leptospira, including CD4+ T cells, which comprised 40% of proliferating cells, compared to 25% in non-infected controls. However, analysis of gene expression did not determine which immunoregulatory pathways were activated. Lymphocytes purified from the lymph node draining the site of colonization, the renal lymph node, also showed an increase in percentage of proliferating B and T cells. However, in contrast to a phenotype of 40% CD4+ T cells in the spleen, the phenotype of proliferating T cells in the renal lymph node comprised 65% CD4+ T cells. These results confirm that the renal lymph node, the local lymphoid organ, is a dominant site containing Leptospira reactive CD4+ T cells and highlight the need to consider the local, vs. systemic, immune responses during renal colonization infection. The use of inbred immunocompetent rats provides a novel tool to further elucidate those pathophysiological pathways that facilitate the unique biological equilibrium observed in reservoir hosts of leptospirosis.

Abnormal Expression of TRAIL Receptors in Decidual Tissue of Chlamydia trachomatis-Infected Rats During Early Pregnancy Loss.

Chlamydia trachomatis is the scientific name of pathogenic bacteria causing infection that has been linked to spontaneous abortion. In this study, the expression pattern of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; a cytokine related to cell apoptosis) and its receptors was monitored in the decidua of C trachomatis-infected pregnant rats during early gestation to investigate the potential role of this molecular system in C trachomatis-induced spontaneous abortion. The data showed that C trachomatis infection significantly altered the messenger RNA (mRNA) expression of the receptors; death receptor (DR) 4 and DR5 increased, but decoy receptor (DcR) 1 and DcR2 decreased. Consistent with mRNA data, immunohistochemical staining of TRAIL and its receptors indicated that both DR4 and DR5 protein levels were elevated in infected tissues, primarily, decidual cells, decidual vessel wall, and uterine glands, whereas DcR1 and DcR2 showed lower levels compared to the noninfected group. Although receptor expression was altered, there was no difference detected in TRAIL expression. The observed altered expression of TRAIL receptors in C trachomatis-infected rats compared to noninfected rats during the embryo implantation phase suggests a possible mechanism for spontaneous abortion due to apoptosis and therefore failed embryo implantation. In addition, the observed increase in caspase-3 levels in infected cells further supports this finding. Taken together, the data presented in this study suggests C trachomatis infection altered the expression of TRAIL receptors, thus representing a general mechanism for C trachomatis-induced spontaneous abortion in C trachomatis-infected rats during early pregnancy loss.

Fasciola hepatica reinfection potentiates a mixed Th1/Th2/Th17/Treg response and correlates with the clinical phenotypes of anemia.

BackgroundFascioliasis is a severe zoonotic disease of worldwide extension caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm and anemia is the main sign. Herein, the profile of the Th1/Th2/Th17/Treg expression levels is analyzed after reinfection, correlating them with their corresponding hematological biomarkers of morbidity.Methodology/Principal findingsThe experimental design reproduces the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 8 weeks (R8), and at 12 weeks (R12), and negative control rats. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. After 20 weeks of primary infection, PI did not present significant changes in the expression of those genes when compared to non-infected rats (NI), but an increase of Il4, Arg1 and Ifng mRNA in the spleen was observed in R12, suggesting the existence of an active mixed Th1/Th2 systemic immune response in reinfection. Foxp3, Il10, Tgfb and Ebi3 levels increased in the spleen in R12 when compared to NI and PI, indicating that the Treg gene expression levels are potentiated in chronic phase reinfection. Il17 gene expression levels in R12 in the spleen increased when compared to NI, PI and R8. Gene expression levels of Il10 in the thymus increased when compared to NI and PI in R12. Ifng expression levels in the thymus increased in all reinfected rats, but not in PI. The clinical phenotype was determined by the fluke burden, the rat body weight and the hemogram. Multivariate mathematical models were built to describe the Th1/Th2/Th17/Treg expression levels and the clinical phenotype. In reinfection, two phenotypic patterns were detected: i) one which includes only increased splenic Ifng expression levels but no Treg expression, correlating with severe anemia; ii) another which includes increased splenic Ifng and Treg expression levels, correlating with a less severe anemia.Conclusions/SignificanceIn animals with established F. hepatica infection a huge increase in the immune response occurs, being a mixed Th2/Treg associated gene expression together with an expression of Ifng. Interestingly, a Th17 associated gene expression is also observed. Reinfection in the chronic phase is able to activate a mixed immune response (Th1/Th2/Th17/Treg) against F. hepatica but T and B proliferation to mitogens is strongly suppressed in all infected rats vs control in the advanced chronic phase independently of reinfection The systemic immune response is different in each group, suggesting that suppression is mediated by different mechanisms in each case. Immune suppression could be due to the parasite in PI and R8 rats and the induction of suppressive cells such as Treg in R12. This is the first study to provide fundamental insight into the immune profile in fascioliasis reinfection and its relation with the clinical phenotypes of anemia.

Transdermal delivery of oleanolic acid attenuates pro-inflammatory cytokine release and ameliorates anaemia in P. berghei malaria

Malaria remains a major health problem in many tropical areas. Severe malaria infection is associated with secondary complications including anaemia leading to a need for the search of affordable antimalarial agents that can clear the parasitaemia and ameliorate anaemia during infection. The current study investigated the effects of transdermally delivered OA on malaria parasites, HCT and selected plasma cytokine concentrations in P. berghei-infected male Sprague-Dawley rats. The study was carried out over a period of 21days, divided into pre-treatment (day 0–7), treatment (day 8–12) and post-treatment (day 13–21) periods. Parasitaemia, HCT, RBC count, Hgb, plasma TNF-α, IL-6 and IL-10 concentrations were monitored in non-infected and infected rats following a once-off application of an OA-pectin patch (34mg/kg). Animals treated with drug-free pectin and CHQ (30mg/kg, p.o) twice daily for 5 consecutive days acted as negative and positive controls respectively. Infected control animals exhibited increased percentage parasitaemia, TNF-α, IL-6, IL-10 and a reduction in HCT. Interestingly, OA-pectin patch application cleared the malaria parasites and increased HCT values back to normalcy. Furthermore, TNF-α, IL-6 and IL-10 were reduced by day 12 of the study. These findings suggest that the OA-pectin patch delivers therapeutic doses of OA which are able to attenuate cytokine release and ameliorate anaemia during malaria infection. Therefore, transdermally delivered OA may be a potent therapeutic agent for malaria and amelioration of anaemia.

The Effects of Transdermally Delivered Oleanolic Acid on Malaria Parasites and Blood Glucose Homeostasis in P. berghei-Infected Male Sprague-Dawley Rats.

The present study investigated the effects of transdermally delivered oleanolic acid (OA) monotherapy and in combination with chloroquine (CHQ) on malaria parasites and glucose homeostasis of P. berghei-infected male Sprague-Dawley rats. Oral glucose test (OGT) responses to OA-pectin patch and CHQ-OA combination matrix patch were monitored in non-infected and infected rats. To evaluate the short-term effects of treatment, percentage parasitaemia, blood glucose, glycogen and plasma insulin were monitored in separate groups of animals treated with either OA-patch monotherapy or CHQ-OA combination pectin patch over a 21-days period. Animals treated with drug-free pectin and CHQ acted as untreated and treated positive controls, respectively. Infected control rats exhibited significantly increased parasitaemia which was accompanied by hypoglycaemia. Both OA monotherapy and CHQ-OA combination therapy reduced and cleared the malaria parasites within a period of 4 and 3 days, respectively. Compared to respective controls groups, OGT responses of animals treated with OA monotherapy or CHQ-OA combination therapy exhibited lower blood glucose levels at all time points. A once-off transdermal application of OA-patch or CHQ-OA combination patch significantly improved blood glucose concentrations inducing any changes in insulin concentration. Transdermal OA used as a monotherapy or in combination with CHQ is able to clear and reduce the malaria parasites within a shorter period of time without eliciting any adverse effects on glucose homeostasis of P. berghei-infected rats.

Regulatory parameters of the lung immune response during the early phase of experimental trichinellosis

Parasitic infection caused by Trichinella spiralis provokes an early stimulation of the mucosal immune system which causes an allergic inflammatory response in the lungs. The present work was intended to characterize the kinetics of emergence of regulatory parameters in Wistar rat lungs during this early inflammatory response, between days 0 and 13p.i. The presence of regulatory cells such as regulatory T cells (Tregs) and alternatively activated macrophages (AAM) was analyzed in lung cell suspensions. Moreover, a regulatory cytokine (TGF-β) was studied in lung tissue extracts. Considering that newborn larvae (NBL) travel along the pulmonary microvasculature, the ability of this parasite stage to modulate the activation of lung macrophages was evaluated. For this purpose, lung macrophages from non-infected or infected rats (day 6p.i.) were cultured with live or dead NBL. Arginase activity (characteristic of AAM) and nitric oxide (NO produced by iNOS, characteristic of classical activated macrophages) were measured after 48h. Our results revealed a significant increase in the percentage of Tregs on days 6 and 13p.i., arginase activity on day 13p.i. and TGF-β levels on days 6 and 13p.i. Lung macrophages from non-infected rats cultured with live NBL showed a significant increase in arginase activity and NO levels. Live and dead NBL induced a significant increase in arginase activity in lung macrophages from infected rats. Only live NBL significantly increased NO levels in these macrophages. The present work demonstrates for the first time, the emergence of regulatory parameters in the early lung immune response during T. spiralis infection. The immumodulatory properties exerted by NBL during its passage through this organ could be the cause of such regulation. Moreover, we have shown the ability of NBL to activate macrophages from the lung parenchyma by the classical and alternative pathways.

A novel soft tissue model for biomaterial-associated infection and inflammation

Event Abstract Back to Event A novel soft tissue model for biomaterial-associated infection and inflammation Sara Svensson1, 2*, Margarita Trobos1, 2*, Maria Hoffman1, 2*, Birgitta Norlindh1, 2*, Sarunas Petronis2, 3*, Jukka Lausmaa2, 3*, Felicia Suska1, 2 and Peter Thomsen1, 2* 1 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Department of Biomaterials, Sweden 2 University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden 3 SP Technical Research Institute of Sweden, Chemistry, Materials and Surfaces - Medical Device Technology, Sweden Introduction: Biomaterial-associated infection is an important complication of medical devices that often requires removal of the implant for successful healing. The mechanisms of bacterial persistence and how the host immune cells are affected by the presence of a colonized biomaterial are incompletely understood. The exploration of novel infection control strategies would be enabled by the development of experimental models allowing the quantitative determination of host-bacteria interactions in vivo. This study describes a soft tissue model for the investigation of bacteriological, morphological and molecular events taking place on the implant surface and in the surrounding exudate and tissue[1]. Materials and Methods: Smooth and nanostructured Ti disks with or without noble metal chemistry (Ag, Au, Pd) and sham sites were inoculated with a dose of 106 CFU Staphylococcus epidermidis (ATCC 35984) subcutaneously on the back of rats. Implant and sham sites were analysed after 4 h, 24 h and 72 h with respect to the number of viable bacteria and the number, viability and gene expression (TNF-α, IL-6, IL-8, IL-10, TLR2, TLR4, elastase) of host cells. The tissue response and the location of S. epidermidis in the interface tissue, were evaluated by histology and FISH, respectively. Non-infected rats were controls. Data was statistically evaluated in PASW/Statistics 18.0 and different tests were applied depending on the type of comparison (n=7-8). The study was approved by the local ethical committee for laboratory animals (Dnr 254/11). Results and Discussion: The results revealed distinct differences between infected and control sites, e.g. in the number, type and viability of host cells surrounding the implant. Host cells at infected sites showed significantly higher gene expression activity of genes related to inflammation, bacterial recognition and tissue degradation than control sites. In the tissue, bacterial cells were primarily located in close proximity to the implant, both extra- and intracellularly (Fig 1). Cytospin preparations of the exudate showed that both mono- and polymorphonuclear cells contained bacterial cells within the cytoplasm (Fig 2). Comparisons between materials revealed elevated cell recruitment, higher neutrophil presence and higher pro-inflammatory gene expression in host cells surrounding noble metal coated Ti, which was in addition associated with a lower number of viable bacteria at 24 h (Fig 3). Interestingly, all implant surfaces harboured less bacteria (ten fold) than the surrounding exudate at all time points. In addition, S. epidermidis still persisted at both material and sham sites after 72 h, indicating a need for longer-term studies. Conclusion: This model allows detailed analysis of early events in inflammation and infection associated to biomaterials in vivo, leading to insights into host defence mechanisms in biomaterial-associated infections. BIOMATCELL Vinn Excellence Center of Biomaterials and Cell Therapy; Swedish Research Council (grant (K2015-52X-09495-28-4); Hjalmar Svensson Research Foundation; IngaBritt and Arne Lundberg Foundation; Felix Neubergh Foundation

Occurrence and identification of hemotropic mycoplasmas (Hemoplasmas) in free ranging and laboratory rats (Rattus norvegicus) from two Brazilian zoos.

BackgroundHemotropic mycoplasmas (hemoplasmas), bacteria belonging to the class Mollicutes, are obligatory red blood cell pathogens of a variety of animal species. They may cause acute anemia that is life-threatening or chronic disease that is clinically silent, but may interfere with results of experimental studies when using infected animals. Since these bacteria cannot be cultivated, molecular techniques are the gold standard for diagnosing an infection, investigating its prevalence, and describing new species. Mycoplasma coccoides and M. haemomuris are the most commonly recognized hemoplasmas in the blood of wild and laboratory rodents. Neither the epidemiology nor clinical and molecular characterization of hemoplasma infection in free-ranging rodents in Brazil has been previously reported. The aims of this study were to investigate the occurrence of hemoplasmas in free-ranging rats (Rattus norvegicus) captured in the Passeio Público and Curitiba Zoo and compare hematologic parameters of infected and non-infected animals.ResultsAnti-coagulated blood samples collected from 43 free-ranging and 20 nursery rats were included in the study. Overall 63.5 % were positive using SYBR® Green quantitative PCR (qPCR) of the 16S rRNA gene to screen for hemoplasma infection (72 % among free-ranging rats; 45 % among laboratory-raised rats). Sequencing of the qPCR products showed that all but one sample had >98 % identity to M. haemomuris. Phylogenetic analysis based on a fragment of approximately 1300 bp of the 16S rRNA gene showed 99 % identity to a new hemoplasma from European rats and 98 % identity to a hemotropic mycoplasma described infecting a European harvest mouse (Micromys minutus). No statistically significant changes in hematologic parameters between infected and non-infected rats were found, confirming the low pathogenicity and/or silent characteristics of the infection.ConclusionsOur findings suggest that hemoplasmas are likely endemic in rodent species in this region. The epidemiology, especially as it relates to the mode of transmission, needs to be further investigated as well as the possibility that other animal species, including humans, might become infected.

A novel soft tissue model for biomaterial-associated infection and inflammation – Bacteriological, morphological and molecular observations

Infection constitutes a major risk for implant failure, but the reasons why biomaterial sites are more vulnerable than normal tissue are not fully elucidated. In this study, a soft tissue infection model was developed, allowing the analysis of cellular and molecular responses in each of the sub-compartments of the implant–tissue interface (on the implant surface, in the surrounding exudate and in the tissue). Smooth and nanostructured titanium disks with or without noble metal chemistry (silver, gold, palladium), and sham sites, were inoculated with Staphylococcus epidermidis and analysed with respect to number of viable bacteria, number, viability and gene expression of host cells, and using different morphological techniques after 4 h, 24 h and 72 h. Non-infected rats were controls. Results showed a transient inflammatory response at control sites, whereas bacterial administration resulted in higher recruitment of inflammatory cells (mainly polymorphonuclear), higher, continuous cell death and higher gene expression of tumour necrosis factor-alpha, interleukin-6, interleukin-8, Toll-like receptor 2 and elastase. At all time points, S. epidermidis was predominantly located in the interface zone, extra- and intracellularly, and lower levels were detected on the implants compared with surrounding exudate. This model allows detailed analysis of early events in inflammation and infection associated to biomaterials in vivo leading to insights into host defence mechanisms in biomaterial-associated infections.